Interactions between molecules are necessary for all bioreactions. The interactions between antibodies and antigens, and ligands and receptors are crucial for initiating a series of pathways to response to external stimuli. Identification of a specific molecule involved in the interactions is very important to research and pharmaceutical development.
For example, after the completion of the human genomic project, most human expressed genes have been identified. However, in the emerging proteomics era, proteins act as key players in the unveiling of these genes. In order to effectively decipher the mystery of these proteins, many efforts have been taken to generate at least one antibody to every human expressed gene in the human genome. Antibodies with their inherent sensitivity and specificity thus become the most versatile tools for providing one-to-one relationship with their target proteins. However, one of the complexities is that these proteins do not always behave in the same way when placed in different (e.g. cancer) cells. Many proteins are known for their translocation capability in different cell contexts; thus the corresponding antibodies will recognize distinct locations in a given cell. Moreover, in many cases, protein translocations are involved in the activation of tumor cells. Detecting the movements might help diagnose the type and stage of cancer in the future. The high-throughput approach with direct selection of antigen on the cell membrane may allow the discovery of potential therapeutic targets and new disease markers.
However, while numerous useful antibodies have been generated, the main limitations are two folds, namely (1) how to directly uncover specific antigens (e.g. membrane or surface markers) in a given cell and (2) how to rapidly identify the valuable antibodies recognizing these antigens.
Taking membrane proteins as an example, many membrane proteins are implicated in particular disease states, such as lung cancer, and often are attractive therapeutic targets. Systematic and quantitative profiling of membrane proteins may facilitate our understanding of their roles in regulating biological processes in various disease states. Approximately 20 to 30% of open reading frames of most sequenced genomes are estimated to encode integral membrane proteins (Blonder, J., et al., Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. Journal of Proteome Research, 2002. 1(4): p. 351-360; Han, D. K., et al., Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nature Biotechnology, 2001. 19(10): p. 946-951). However, the membrane proteome has not been mapped and is experimentally challenging because of the low abundance of membrane proteins. Moreover, using high-throughput proteomics and microarray-based screening studies, the identified novel targets often lack antibodies for characterization of their location and function, further preventing researchers from pursuing potential membrane proteins. A direct expression profiling method for identifying antibodies and recognizing receptors (surface markers or membrane-associated proteins) is essential.
Random screening is time-consuming and usually impracticable due to the various physiological conditions. Using individual antibodies to screen new receptors is impossible due to the requirement of more than 25000 antibodies to cover the whole genome. Moreover, cancer cells often have multiple overexpressed receptors and distinct cell types that may have different location profiles for the same target protein. Therefore, it is necessary to develop a rapid and effective method to address these questions at the same time.